1. Maximum esterase production is observed at the early stationary phase of growth.
2. Other methods to lyse cells can be used, such as lysozyme treatment with sonication, or pressure techniques.
3. The esterase system is intracellular in LAB
4. The electrophoretic reagents must be of high quality.
5. The high-molecular-weight proteins (>100 kDa) are better resolved on 7-8% acrylamide gels. We obtain better results with 12% acrylamide gels.
6. Band resolution is improved if small volumes of samples are applied to the gels.
7. Electrophoresis can be run at 4°C to avoid enzyme denaturalization.
8. The disadvantage of this technique is that a substrate of a-NA fatty acid derivatives longer than propionate may not be easily accessibile through the net of the electrophoresis gel, lending one to the false conclusion that there is no activity (6).
9. Fast Red TR forms a complex red color that precipitates, but the bands of activity can be noticable.
10. Fast Blue can be used as a substrate to develop color. In this case a complex dark blue color is formed.
11. The electrophoretic protein patterns provide a reliable way to differentiate strains and reveal the complexity of esterase systems (7).
1 Bornscheuer, U. T. (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol. Rev. 26, 73-81.
2. Tsakalidou, E., Zoidou, E., and Kalantzopoulos, G. (1992) Esterase activities of cell-free extracts from strains of Lactococcus lactis subsp. lactis isolated from traditional Greek cheese. J. Dairy Res. 59, 111-113.
3. Vafopoulou-Mastrojiannaki, A., Litopoulou-Tzanetaki, E., and Tzanetakis, N. (1996) Esterase activities of cell-free extracts from wild strains of leuconostoc and heterofermentative lactobacilli isolated from traditional Greek cheese. Lett. Appl. Microbiol. 23, 367-370.
4. De Man, J. C., Rogosa, M., and Sharpe, M. E. (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135.
5. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685.
6 Morichi, T., Sharpe, M. E., and Reiter, D. (1968). Esterase and other soluble proteins of some lactic acid bacteria. J. Gen. Microbiol. 53, 405-414
7. Katz, M., Medina, R., Gonzalez, S., and Oliver, G. (2002) Esterolytic and lipolytic activities of lactic acid bacteria isolated from ewe's milk and cheese. J. Food Prot. 65, 1997-2001.
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