Site-Directed Mutagenesis Revealed Role of Subsite Residues of Mucor Pusillus Pepsin in Catalytic Function

  • Young-Nam Park
  • Makoto Nishiyama
  • Sueharu Horinouchi
  • Teruhiko Beppu
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 362)


A few aspartic proteinases, chymosin obtained from calf stomach1 and Mucor pusillus pepsin (MPP) produced by a filamentous fungus Mucor pusillus,2 have been used as the milk-clotting enzymes for industrial production of cheese. Milk-clotting by the aspartic proteases is caused by the specific cleavage of κ-casein, which functions as a stabilizer of milk micelle, at the peptide bond of Phe 105-Met 106. Chymosin and MPP are discriminated from other members of the aspartic proteinases by high selectivity to the peptide bond of Phel05-Metl06 causing destabilization of milk micelles, along with very weak activity of non-specific proteolysis, which assure the production of the clotted materials in a high yield. In the last several years we have constructed an expression system for MPP using Sac-charomyces cerevisiae as a host,3 in which the proMPP secreted into the medium is converted to the mature protease through the self-processing activity as well as by the host-dependent cleavage.4 This system possesses an inherent advantage, which enables us to obtain even inactive mutant enzymes in mature forms. By using this yeast expression system, we conducted site-directed mutagenesis of MPP, especially on the amino acid residues involved in substrate binding.


HPLC Electrophoresis Assure Oligosaccharide Galactose 


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Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Young-Nam Park
    • 1
  • Makoto Nishiyama
    • 1
  • Sueharu Horinouchi
    • 1
  • Teruhiko Beppu
    • 1
  1. 1.Department of Agricultural Chemistry Faculty of AgricultureThe University of TokyoTokyoJapan

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