Evidence for proteolytic activity of Lactobacilli isolated from kefir grains

Biotechnology & Biotechnological Equipment

Subject: Biotechnology
Publisher: Diagnosis Press
ISSN 1310-2818

Volume 20, Number 2
Date: June 2006


Evidence for proteolytic activity of Lactobacilli isolated from kefir grains

P. Kabadjova-Hristova, S. Bakalova, B. Gocheva and P. Moncheva

Sofia University, Biological Faculty, Department of Microbiology, Sofia, Bulgaria


The proteolytic activity of 276 natural isolates from kefir grains was investigated. Evidence for extracellular proteolytic activity was demonstrated for 49 Lactobacillus strains. One strain, Lactobacillus kefir DR22x, was selected as a producer of proteinases. Lactobacillus kefir DR22x strain produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+– free buffer. The crude proteinase extract showed the highest activity at pH 7.2 and 37 ºC. The proteolytic activity was shown to be maximal in the late exponential growth. A study using several protease inhibitors suggested that this activity is associated with serine-type proteases. Considering the substrate specifity, the enzyme is similar to the lactococcal P1–type proteinases, since it completely hydrolysed only β-casein, showing very low activity towards α-casein.


  1. El Soda M., Desmazeaud M.J., Le Bars D., Zevaco C. (1986) J. Food Prot., 49, 361-365.
  2. Fira D., Kojic M., Banina A., Spasojevic I., Strahinic I., Topisirovic L. (2001) J. Appl. Microbiol., 90, 123-130.
  3. Khalid N., Marth E.H. (1990) J. Dairy Sci., 73, 3068-3076.
  4. Kojic M., Fira D., Banina A., Topisirovic L. (1991) Appl. Envron. Microbiol., 57, 1753-1757.
  5. Kunji E.R.S., Mierau I., Hagting A., Poolman B., Konings W.N. (1996) Ant. van Leew., 70, 187-221.
  6. Kunitz M. (1946) Gen. Physiol., 30, 291-310.
  7. Laemmli U.K. (1970) Nature, 227, 680-685.
  8. Pereira C.I., Barreto Crespo M.T., San Romao M.V. (2001) Int. J. Food Microbiol., 68, 211-216.
  9. Rimada P.S., Abraham A.G. (2001) J. Dairy Res., 68, 653-661.
  10. Takizawa S., Kojima S., Tamura S., Fujinaga S., Benno Y., Nakase T. (1994) Int. J. of Syst. Bacteriol., 44, 435-439.
  11. Tsakalidou, Anastasiou R., Vandenberghe I., Van Beeumen J., Kalantzopoulos G. (1999) Appl. Environ. Microbiol., 65, 2035-2040.
  12. Yüksekdag Z.N., Beyath Y., Aslim B. (2004) Nahrung/Food, 48, 218-220.