Purification and properties of intracellular esterases from Streptococcus thermophilus

Two of the three intracellular esterases identified in Streprococcus thermo philus were purified to homogeneity using ammonium sulphate fractionation a nd three chromatographic steps: anion exchange, hydrophobic interaction and gel filtration. The subunit molecular masses of esterases I and II were si milar to 34 and similar to 60 kDa, respectively. The holoenzyme molecular m asses of esterases I and II were similar to 50 and similar to 60 kDa, respe ctively, indicating that esterase I could be a dimer and that esterase II w as a monomer. Phenylmethylsulphonyl fluoride inhibited the activity of both esterases, but to different degrees. Dithiothreitol, N-ethylmaleimide and EDTA strongly inhibited the activity of esterase I but significantly enhanc ed the activity of esterase II. Esterase I was active on p-nitrophenyl este rs of the short-chain fatty acids from C-2 to C-10 and esterase II was acti ve on p-nitrophenyl esters of the C-2-C-6 fatty acids. For both enzymes, ma ximum activity was obtained with p-nitrophenyl butyrate (C-4). The K-m valu es of esterase I on p-nitrophenyl esters of C-2-C-8 fatty acids ranged from 6.7 to 0.004 mM and the corresponding V-max values ranged from 8.12 to 1.1 2 mu mol min(-1) mg(-1) protein. The N-terminal amino acid sequences of the two esterases also differed. The major esterase (I), accounting for simila r to 95% of the total esterase activity, was further characterized. Esteras e I was also active against tributyrin (C-4), dicaproin (C-6) and monoglyce rides of up to C-14 with maximum activity on monocaprylin (C-8). Decreasing ph (from 8.0 to 5.5), temperature (from 37 degrees to 25 degreesC) or wate r activity (from 0.99 to 0.80) considerably reduced the activity of esteras e I, whereas increasing NaCL concentration up to 7.5% (w/v) markedly enhanc ed the activity of this enzyme. Esterase I may play a role in the developme nt of cheese flavour with respect to lipolysis. (C) 2001 Elsevier Science L td. All rights reserved.