Expression of buffalo chymosin in Pichia pastoris for application in mozzarella cheese

Chymosin, a major component of rennet, is used extensively as a milk clotting enzyme in cheese making industry. Increased demand coupled with scarce availability of native animal rennet has encouraged use of recombinant chymosin (rChymosin) in the dairy industry. Better stability of buffalo chymosin as compared to its other sources such as lamb, calf etc. makes it a desirable source for recombinant enzyme production. In the present investigation, we report the inducible expression of recombinant buffalo prochymosin in P. pastoris, its chromatographic purification and mozzarella cheese preparation. RNA extracted from the abomasum of a suckling calf buffalo was used to synthesize prochymosin cDNA and expressed in P. pastoris X-33 as well as SMD1168. We could observe 68% higher expression of recombinant buffalo chymosin in X-33 in comparison to SMD1168 strain. The enzyme was subsequently purified to approximately 28 fold. The new recombinant yeast strain could represent a novel and excellent source of high level expression of chymosin for application in cheese making industry.