Intracellular esterase from Lactococcus lactis subsp. lactis NCDO 763: Purification and characterization
Chich, J.-Francois; Marchesseau, K.; Gripon, J.-Claude
International Dairy Journal 7(2-3): 169-174
The presence of esterolytic activities in the intracellular extract of Lactococcus lactis subsp. lactis NCDO 763 was investigated. One major activity hydrolyzing beta-naphthyl butyrate was detected. This activity was purified to homogeneity. The enzyme was a homotrimer; the molecular mass of the monomer was estimated by SDS-PAGE to be 29 kDa; by mass spectrometry, it was 29655 +- 30 Da. The pI of the molecule was 4.5. The enzyme was active on para-nitrophenyl esters from C2 to C12 and on orthonitrophenyl butyrate, with a maximum activity on p-nitrophenyl butyrate. The optimum activity on this substrate was found at pH 8.0 and at 55 degree C; kinetic parameters for this substrate were measured at 55 degree C and were K-M = 0.11 mM and V-max = 8.17 mu-mol min-1. The enzyme was strongly inhibited by Pefabloc, diisopropyl fluoro-phosphate and 3,4-dichloroisocoumarin, demonstrating that it was a serine esterase. The amino-terminus of the enzyme was sequenced. The role of this enzyme in cheese ripening is discussed.