Sirma Yegin

• This project explored the suitability of the aspartic proteinase from Mucor mucedo DSM 809 as novel microbial rennet. The native proteinase was produced via submerged cultivation in shaking flasks. Under optimized conditions, the peak of production (130 U/ml) was observed after 48 h of cultivation at 24°C and 220 rpm. The chromatographically purified enzyme depicted a molecular weight (MW) of 32.7 kDa, an isoelectric point (pI) value of 4.29, and no evidence of N-linked glycosylation was found. When acting on milk to provoke curd formation, the M. mucedo DSM 809 proteinase showed maximum potency at pH 5.0 and at 40°C. The enzyme was heat-sensitive and became completely inactivated after incubation at 55°C for 10 min. The cDNA encoding the aspartic proteinase of M. mucedo has been identified (GenBank accession # JN660818). The proteinase gene was cloned into pGAPZαA vector and expressed in Pichia pastoris X-33. Maximum enzyme production was observed when initial medium pH was 3.5 at 20°C and utilizing 4% glucose as a carbon and energy source. The enzyme was secreted in glycosylated form; two potential N-glycosylation sites were elucidated by site directed mutagenesis. All of the generated mutants have exhibited increase in milk clotting activity. The maximum activity obtained was 624 U/ml. Effect of copy number of the expression cassette on the activity has been discussed. Thermal stability studies of the purified proteins revealed that there was almost no effect of glycosylation on the thermal stability of the enzymes. The ratio of milk clotting activity to proteolytic activity for the unglycosylated enzymes was almost 3-fold higher than the glycosylated counterparts. The M. mucedo proteinase / P. pastoris expression system has potential for the industrial production of a novel milk-clotting enzyme for cheese manufacturing.