Ultrasound Treatment for Harvesting an Aminopeptidase from Lactic Acid Bacteria and Quantitation of the Enzyme by Enzyme-Linked Immunosorbent Assays

Abstract

Ultrasound treatment of Lactococcus lactis subsp. cremoris AM2 was optimized to release a maximum amount of intracellular aminopeptidase without modifying the antigenicity of the enzyme. The cells were sonicated three times for 30 s at 23 W. Antibodies produced against the aminopeptidase purified from L. lactis subsp. cremoris AM2 enabled us to use immunoblotting to detect the enzyme in the lysates of all of the lactococci tested but not in the lysates of Leuconostoc strains, lactobacilli, and Streptococcus salivarus subsp. thermophilus . A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the purified aminopeptidase; the detection limit was 4 ng/ml. The aminopeptidase in the supernatant obtained after the ultrasound treatment of strain AM2 cells was detected with the ELISA starting with a total protein concentration of 200 ng/ml. The proportion of equivalent purified aminopeptidase in the supernatant of L. lactis subsp. cremoris AM2 was about 2% of the total protein. Similarly, the aminopeptidase was quantified in different lactococci; the percentages varied between 0.16 and 2%, depending on the strain. The aminopeptidase content in a mixture of several lactic bacteria was also determined with the sandwich ELISA.

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

“…The authors suggest that this may be similar to the PepC enzyme described from Lactococcus lactis subsp, cremoris AM2 [78,79]. However, antibodies raised to the lactococcal aminopeptidase C did not react with extracts from lactobacilli, leuconostocs or S. thermophih~s [161]. So far there have been no reports of an aminopeptidase A from lactic acid bacteria other than lactococci.…”