Expression and characterization of recombinant chymosin in Pichia pastoris

Chymosin is a proteolytic enzyme that coagulates milk efficiently and widely used in cheese industry. It obtained from fourth stomach of nerwborn ruminants. But its quality and quantitiy can not meet demand of the market. Recombinant chymosin is the best alternative for cheese industry. Present day high quantitiy of chmosin can be produced with recombinant enzyme technology. Until now, bovine; camel; goat; buffalo were used as a gene sources for chymosin expression. In the present study, yak (Bos grunnniens) prochymosin gene was used as gene source and recombinant chymosin was expressed in P. pastoris. Expression casettes have Saccharomyces cerevisiae α-mating factor (α-MF), HSA (Human Serum Albumin), PIR1 (P. pastoris Protein with Internal Repeats) ve PHO1 (P. pastoris Acid Phosphatase) signal sequences were constructed to observe impact of secretion signals on expression of enzyme. Active chymosin expression was achieved into supernatant under the control of methanol-inducible AOX1 promoter. Trials continued in bioreactor only expression casette which have α-MF secterion signal. Recombinant chymosin production was analyzed in flusk cultures then continued with the bioreactor which included basal salt medium. Eznyme characterization was performed with raw supernatant obtained from bioreactor. Present study will contribute to literature about recombinant chmosin which its gene source is yak.