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Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange
J R Broadbent1, B T Rodríguez, P Joseph
1Western Dairy Center and Department of Nutrition and Food Sciences, Utah State University, Logan, UT, USA.
Journal of Applied Microbiology
|May 16, 2006
Summary
Genetic modification of Lactococcus lactis lactocepin (prtP) altered enzyme specificity to reduce bitter flavor without impacting essential strain functions. This research offers a method for improving starter cultures.
Area of Science:
- Food Microbiology
- Enzymology
- Genetic Engineering
Background:
- Lactocepin, encoded by prtP genes in Lactococcus lactis, can produce bitter off-flavors in dairy products.
- Modifying lactocepin specificity is crucial for enhancing the sensory quality of fermented dairy products.
Purpose of the Study:
- To investigate if altering lactocepin substrate binding regions via gene replacement can change enzyme specificity in Lactococcus lactis.
- To assess if these modifications affect critical starter culture properties like milk acidification and autolysis.
Main Methods:
- Employed a two-step gene replacement strategy to convert bitter (group h) lactocepin determinants to non-bitter (group b) variants in L. lactis prtP genes.
- Analyzed lactocepin activity and specificity using reversed-phase high-pressure liquid chromatography on alpha(s1)-casein fragments.
- Evaluated milk acidification properties and autolysis propensity of modified strains.
Main Results:
- Gene replacement successfully altered lactocepin specificity, consistent with predicted amino acid changes in substrate-binding regions.
- Some modified strains exhibited no statistically significant difference in milk acidification compared to wild-type strains.
- Autolysis propensity remained largely unchanged in the engineered L. lactis derivatives.
Conclusions:
- Allele exchange in lactocepin substrate binding regions effectively modifies enzyme specificity in industrial L. lactis strains.
- The developed methodology can be applied to commercial starter cultures to prevent bitter flavor defects.
- Engineered prtP derivatives are suitable for commercial application in dairy fermentation.
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