A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris.
The main autolysin PA49.5, an enzyme that hydrolyzes or destroys the components of a biological endogenous cell or a tissue, was purified 3045 times from the homogenate of a whole cell extract of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5), with a recovery yield of 52%. The purification of the protein was carried out through a micro-purification technique using SDS-BigCHAP polyacrylamide gel electrophoresis and concentrated with a Microcon-10 filtration system. SDS-polyacrylamide gel electrophoresis of the purified enzyme confirmed the presence of only one band having a molecular weight of 49.5 kDa. In view of its insolubility, PA49.5 contained in the cell extract precipitate was solubilized in the presence of 0.1% (w/v) of BigCHAP, a non-ionic detergent. Higher concentrations of this detergent completely inhibited the activity of solubilized PA49.5 or prevented its solubilization. The optimal pH and temperature for PA49.5 enzymatic activity are 7.5 and 45 degrees C respectively. In addition 0.1% or less of PA49.5 significantly increased Mc5 lysis. We observed 55% more lysis with 0.25 mug of purified PA49.5 compared to the control. Gas chromatography analysis of the components of the crude cell extract, of the precipitate and of the supernatant indicates the presence of at least 6 fatty acids. The long-chained fatty acids (e.g. C18:0 and C18:3) detected represent 81.65% of the precipitate from which PA49.5 was purified. Of these two acids, the C18:0 (stearic acid) alone represents 47.40% of the precipitate. Mc5 releases proteins at the beginning (major peak) and at the end (moderate peak) of the exponential stage of growth. Analysis by denaturing polyacrylamide gel electrophoresis with Mc5 cell walls incorporated as the autolysin's substrate identified a band corresponding to PA49.5 in the second peak of protein secretion.
Dako
Etienne
E
Ecole des sciences des aliments, de nutrition et d'études familiales, Faculté des sciences de la santé et des services communautaires, Université de Moncton (N.-B) Canada, E1A 3E9. etienne.dako@umoncton.ca
Jankowski
Christopher K
CK
Bernier
Anne-Marie
AM
Asselin
Alain
A
Simard
Ronald E
RE
D001329:Autolysis; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005227:Fatty Acids / Q000032:analysis*; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology*; D008970:Molecular Weight; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000302:isolation & purification*; D019936:Probiotics; D012995:Solubility; D013696:Temperature
D005227:Fatty Acids; D009238:N-Acetylmuramoyl-L-alanine Amidase