Molecular Modification Of Recombinant Mucor Pusillus Chymosin And Optimization Of Fermentation Conditions For Chymosin Production

Cheese is a new growth point in China’s dairy industrial development, butChymosin used in cheese production is in a growing shortage, so Genetic engineeringchymosin with low cost and short production cycle has been widely valued. However,our research started relatively late in this area. Therefore, the study of geneticengineering chymosin is of great importance. This study aims to transform recombinantMucor pusillus chymosin in molecular with biotechnology in order to get the mutantstrain of high milk clotting activity、low proteolytic activity and low thermal stability.This paper mainly studies the recombinant Mucor pusillus chymosin from threeaspects; first of all, chose the mutant site for Mucor pusillus chymosin genetransformation and expression in Pichia pastoris. Secondly, the purification andproduction of before and after modification chymosin, comparison between theenzymatic properties of chymosin was made. Finally, the optimal chymosin fromexcellent mutants strain producing conditions was studied. Specific studies andconclusions as follows:(1) The Mucor pusillus chymosin gene at positions218and287of the proteinproduct are replaced. The Thr(ACC) of218gene was mutated into Ala (GCT)(T218A)and Ser (TCC)(T218S), the Leu (CTT) of287gene was mutated into Gln(CAA)(L287G);The mutant gene of chymosin was transferred into Pichia pastoris. Themilk clotting activity of218site-directed mutant chymosin was significantly increased.T218A, T218S, and L287G mutation reduced proteolytic activity of chymosin.T218Smutant strains chymosin was screened and had higher milk clotting activity, lowerproteolytic activity and lower thermal stability.(2) The preparation technology research on chymosin before and aftermodification. Before and after modification chymosin were purified by using ethanolprecipitation、size-exclusion chromatography using Sephadex G-75and ion-exchangechromatography using DEAE-52cellulose. The purification process and purificationstep were simple and possess good reproducibility.(3) Comparisons were made about the chymosin before and after modification.The C/P ratio of mutant Chymosin rose by2.27times, The relative milk clotting activitywas about40%when treated at45℃for120min. (4) The properties of chymosin before and after modification were studied. Theoptimal temperature of the chymosin was60℃, the mutant chymosin was50℃, It’smore suitable for cheese production. The optimal pH of the chymosin was5.5, stabilityin pH6.0.Different CaCl2concentrations, different concentrations of Na+, and effects ofmetal ions on the milk clotting activity were substantially the same curd. Experimentsshowed that enzymatic properties were the same to expected results.(5) Fermentation condition optimization of excellent mutant strain that containsmutational chymosin gene. Study the T218S mutant strain fermentation conditions. Milkclotting activity can be as an indicator.By single factor test, the fermentation time,methanol concentration, inoculum amount and loading volume will impact the milkclotting activity. Optimized by orthogonal experimental conditions for enzymeproduction, the results showed: The optimized parameters of these factors were192h,0.75%/24h,0.4OD600nm,20%.Milk clotting activity reached158.34U/ml.