Purification and properties of intracellular esterases from Streptococcus thermophilus

Two of the three intracellular esterases identified in Streprococcus 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 similar to 34 and similar to 60 kDa, respectively. The holoenzyme molecular masses of esterases I and II were similar to 50 and similar to 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 C-2 to C-10 and esterase II was active on p-nitrophenyl esters of the C-2-C-6 fatty acids. For both enzymes, maximum activity was obtained with p-nitrophenyl butyrate (C-4). The K-m values 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 1.12 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 similar to 95% of the total esterase activity, was further characterized. Esterase I was also active against tributyrin (C-4), dicaproin (C-6) and monoglycerides 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 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. (C) 2001 Elsevier Science Ltd. All rights reserved.