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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.
Abstract:
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.
Insights
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.