Production Of Recombinant Bovine Chymosin By Picha Pastoris And Its Application In Cheese Processing

Compared with chymosin from other origins, transgenetic chymosin has a relatively lower price and higher output with many cheesemaking-suitable traits.The recombinant enzyme has been widely used in cheese factory in many countries. Using the poly-electrotransformation technology,we obtained a recombinant Pichia pastoris strain with multiple copies of bovine chymosin genes. The expression capability of this strain was 2.7fold of the single copy strain, and the Southern Blot analysis showed the copy number of the strain was steady after being inuced for 24h,48h,72h,96h with methanol. With a 5L fermenter, the highest milk clotting activity in the fermentation liqor was obtained at 152SU/mL by adding lOg/L sorbitol at the beginning of introduction phase, oleic acid to 0.2%, 10.0g/L peptone every 12h during induction phase, continuously feeding methanol to keep its concentration at 0.1% and stabilizing the pH at 3.0 with aqueous ammonia. The recombinant chymosin was purified with the phenol sepharose at 2.5mL/min flow rate and 2mol/L ion concentration of the fermentation supernant and the enzyme was recovered at 82.33%.Studies on the characteristics of the recombinant chymosin showed the optimal milk-coagulating temprature was 45℃, at 55℃for 6mins the enzyme lost its activity, the enzyme was stable at pH4.0-7.0 and the highest milk clotting activity was achieved at pH5.0. The above results together with the data of rheological experiments confirmed the recombinant chymosin had similar characteristics to the commercial chymosin. Using recombinant chymosin to make cheddar cheese,we found the output was slightly higher than those made with commercial enzyme but the flavor and mouthfeel were very similar during the ripening period.