Site-Directed Mutagenesis Revealed Role of Subsite Residues of Mucor Pusillus Pepsin in Catalytic Function
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.
KeywordsHPLC Electrophoresis Assure Oligosaccharide Galactose
Unable to display preview. Download preview PDF.
- 1.A.G. Mackinlay, and R.G. Wake, κ-Casein and its attack by rennin (chymosin), in Milk Proteins” vol. 2, H.A. Mckenzie, ed., Academic press, New York, pp. 175–215 (1971)Google Scholar
- 2.K. Arima, S. Iwasaki, and G. Tamura, Milk clotting enzyme from microorganisms, Agrie. Biol. Chem. 31:540–545 (1967)Google Scholar
- 6.J. Aikawa, R. Hiramatsu, M. Nishiyama, S. Horinouchi, and T. Beppu, A yeast expression system and site-directed mutagenesis of a fungal aspartic proteinase, Mucor rennin, in “Structure and Function of the Aspartic Proteinases” B. M. Dunn, ed., Plenum Press, New York, pp. 233–242 (1991)CrossRefGoogle Scholar