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Engineering, expression, purification, and production of recombinant thermolysin
Kuniyo Inouye1, Masayuki Kusano, Yasuhiko Hashida
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
Biotechnology Annual Review
|September 19, 2007
Summary
This review covers recombinant thermolysins, focusing on protein engineering for enhanced activity in synthesizing aspartame precursors. It details methods for expression, purification, and production of this key industrial enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Thermolysin is a thermostable zinc metalloproteinase from Bacillus thermoproteolyticus Rokko, discovered in 1962.
- It's a model enzyme for studying catalytic mechanisms, thermostability, and halophilicity.
- Thermolysin is crucial in industry for synthesizing N-carbobenzoxy L-Asp-L-Phe methyl ester (ZDFM), an aspartame precursor.
Purpose of the Study:
- To review engineering strategies for recombinant thermolysins.
- To outline methods for expression and purification of thermolysin variants.
- To discuss the production of genetically engineered thermolysins with improved ZDFM synthesis activity.
Main Methods:
- Recombinant DNA technology for protein engineering.
- Expression systems for thermolysin production.
- Purification techniques for isolating recombinant enzymes.
Main Results:
- Various strategies for expressing and purifying thermolysin have been successfully developed.
- Genetic engineering approaches aim to enhance thermolysin's activity and properties.
- Recombinant thermolysins offer potential for improved industrial applications.
Conclusions:
- Recombinant thermolysin engineering is vital for optimizing industrial enzyme applications.
- Advances in expression and purification facilitate the production of tailored thermolysin variants.
- Further research in protein engineering can lead to more efficient aspartame precursor synthesis.
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