A Pichia pastoris fermentation process to express Mucor pusillus rennin
Abstract
Rennin from Mucor pusillus (MPR) is an efficient substitute of bovine rennin used as milk clotting enzyme for cheese manufacture. A new fermentation process in the laboratory scale was developed to produce MPR by means of the strain RP61 Muts from Pichia pastoris containing multiple copies of the gene for MPR expression. This process includes three phases, the first one in batch mode for growth on glycerol with MYPG medium supplemented with 10 g/L peptone and 20 g/L yeast extract, the second one a fed-batch phase with glycerol; and the last one with methanol for protein expression. Growth, yield of MPR and milk clotting activity were used to define the best conditions achieved with a methanol feed flow of 4.5 g/L·h, pH of 5.5 at 28 °C. As a result, the yield obtained for MPR was 0.95 g/L at 112 hours of fermentation with milk clotting activity of 565 IU/mL. On the other hand, the volumetric productivity obtained for rennin production was 8.5 mg/L·h, which means 1.7 times higher than those obtained in other works before.
