Jiang Yuanyuan

Screening and Characterization of a Mutant Fungal Aspartic Proteinasefrom

In this study, site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet (MPR) in order to find a potential substitution of commercial chymosin. Mutant G186D/E13D screened from thousands of mutant showed a significant milk-clotting activity (MCA). Mutant G186D/E13D rennet was purified and characterized. The molecular weight was estimated to be 44 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pH (5.0-7.0) and 60oC. The enzyme was inhibited by metal ions (Fe 2+ , Fe 3+ , Cu + and Zn 2+ ), 1.10-Phenantrolin and pepstatin A. Further texture analysis of cheddar cheeses made by non-mutant rennet, mutant (G186D/E13D) rennet and commercial rennet suggested that the soluble nitrogen content and hardness of cheddar cheese made by chimeric mutant rennet was decreased without significantly change in flavor between these cheeses. The result implicated that, in some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably to be candidate for a useful milk coagulant.