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Mutation of a fungal aspartic proteinase, Mucor pusillus rennin, to decrease thermostability for use as a milk
T Yamashita1, S Higashi, T Higashi
1Tokyo Research Laboratory, Meito Sangyo Co. Ltd., Japan.
Journal of Biotechnology
|January 15, 1994
Summary
Mutagenesis of Mucor pusillus rennin (MPR) yielded a mutant enzyme with reduced thermostability, ideal for cheese production. Specific mutations, particularly Gly186Asp, significantly decreased heat stability while maintaining activity.
Area of Science:
- Enzymology
- Molecular Biology
- Food Science
Background:
- Mucor pusillus rennin (MPR) is an aspartic proteinase used as a milk coagulant.
- Decreased thermostability of MPR is desirable for efficient cheese manufacturing.
- Protein engineering can modify enzyme properties for industrial applications.
Purpose of the Study:
- To generate mutant MPR enzymes with reduced thermostability.
- To identify specific mutations responsible for decreased heat stability.
- To assess the utility of engineered MPR for cheese production.
Main Methods:
- Mutagenesis of the Mucor pusillus fungus to create mutant strains.
- Cloning and expression of mutant mpr genes in Saccharomyces cerevisiae.
- Site-directed mutagenesis to confirm residue involvement in thermostability.
Main Results:
- Isolated a mutant strain producing MPR with significantly reduced thermostability.
- Identified two key mutations: Ala101Thr and Gly186Asp.
- The Gly186Asp mutation markedly decreased thermostability; Gly186 is crucial for enzyme conformation.
- A double mutant (Ala101Thr and Gly186Asp) exhibited the lowest thermostability without compromising activity or clotting/proteolytic ratio.
Conclusions:
- Mutagenesis of MPR can effectively reduce thermostability for industrial applications.
- The Gly186 residue plays a critical role in maintaining MPR's thermal stability.
- Engineered MPR variants offer potential for improved cheese manufacturing processes.