Purification and properties of intracellular esterases from Streptococcus thermophilus

Abstract

Two of the three intracellular esterases identified in Streptococcus thermophilus were purified to homogeneity using ammonium sulphate fractionation and three chromatographic steps: anion exchange, hydrophobic interaction and gel filtration. The subunit molecular masses of esterases I and II were ∼34 and ∼60 kDa, respectively. The holoenzyme molecular masses of esterases I and II were ∼50 and ∼60 kDa, respectively, indicating that esterase I could be a dimer and that esterase II was 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 enhanced the activity of esterase II. Esterase I was active on p-nitrophenyl esters of the short-chain fatty acids from C2 to C10 and esterase II was active on p-nitrophenyl esters of the C2–C6 fatty acids. For both enzymes, maximum activity was obtained with p-nitrophenyl butyrate (C4). The Km values of esterase I on p-nitrophenyl esters of C2–C8 fatty acids ranged from 6.7 to 0.004 mm and the corresponding Vmax values ranged from 8.12 to 1.12 μmol min−1 mg−1 protein. The N-terminal amino acid sequences of the two esterases also differed. The major esterase (I), accounting for ∼95% of the total esterase activity, was further characterized. Esterase I was also active against tributyrin (C4), dicaproin (C6) and monoglycerides of up to C14 with maximum activity on monocaprylin (C8). Decreasing pH (from 8.0 to 5.5), temperature (from 37° to 25°C) or water activity (from 0.99 to 0.80) considerably reduced the activity of esterase I, whereas increasing NaCl concentration up to 7.5% (w/v) markedly enhanced the activity of this enzyme. Esterase I may play a role in the development of cheese flavour with respect to lipolysis.