Mutagenesis of a fungus Mucor pusillus, a producer of an aspartic proteinase named Mucor pusillus rennin (MPR), was performed to obtain the mutated enzymes with decreased thermostability, which is desirable for practical use of the enzyme as a milk coagulant for cheese manufacturing. A fungal mutant strain which produced the mutant enzyme with distinctly reduced thermostability was isolated. Two different mutant alleles of the mpr gene, one with a single amino acid exchange of Ala101 for Thr and the other of Gly186 for Asp, were cloned out of this mutant strain. The mutated mpr genes were expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter to produce the active enzymes in extracellular medium. Both of the mutations, especially Gly186Asp, were confirmed to cause a marked decrease in thermostability of the enzyme. All mutants possessing exchanges of Gly186 for various amino acids by site-directed mutagenesis showed a decrease in thermostability, indicating involvement of this residue to maintain a conformation of the enzyme. A double mutant having the both exchanges, Ala101Thr and Gly186Asp, in a single molecule showed the lowest thermostability without decrease in the enzymatic activity as well as the relative ratio of clotting to proteolytic activity.