P np octanoate

Standard conditions: unless otherwise indicated, MZ0003 activity was measured at 405 nm for 20 minutes in a spectrophotometer (SpectraMax Me2, Microplate reader, Molecular Devices, Sunnyvale, CA, USA). Reaction mixture (100 μl) contained 3.5 μg of enzyme, 0.1 M Tris-HCl buffer (pH 8.0) and 1 mM p-NP acetate (Sigma) as substrate dissolved in 100% acetonitrile. Enzymatic activity was assayed in triplicate with an appropriate blank for the correction of the auto hydrolysis of the substrate. One unit of enzymatic activity was defined as the amount of protein that released 1 μmol of p-nitrophenoxide/min from p-NP esters. Molecular coefficient extinction of 18,000 M^-1 cm^-1 was used for the calculation.
MZ0003 esterase activity was determined towards the acyl chain length of different p-NP esters, dissolved in 100% acetonitrile and purchased from Sigma-Aldrich: p-NP acetate, p-NP butyrate and p-NP octanoate, in the standard assay conditions mentioned in the enzyme activity assay paragraph.
Kinetic parameters were calculated for p-NP acetate using a concentration range of 0 to 4.5 mM in presence of 10 μg of enzyme and 0.1 M Tris-HCl pH 8.0. Initial velocities were calculated with the SoftMax Pro software (Molecular Devices) and then data were fitted with the enzyme kinetic analysis module of Sigmaplot 12.5 (Systat Software, Inc., San Jose, CA).

The standard reaction was carried out with the appropriate amount of purified PE8 or its mutants in 1 ml mixtures containing 100 mM Tris-HCl (pH 7.5) buffer and 1 mM p-NP acetate (Sigma-Aldrich, Milwaukee, WI, USA, dissolved in acetonitrile)^{18 (link)}. The activities were determined at 30 °C and 405 nm using a DU800 UV/Visible spectrophotometer (Beckman, Houston, TX, USA). All experiments were performed in triplicate and corrected for substrate autohydrolysis. Substrate specificity assays were performed with p-NP acetate, p-NP butyrate (Sigma-Aldrich), p-NP hexanoate (TCI, Tokyo, Japan) and p-NP octanoate (Sigma-Aldrich).
The kinetic parameters were obtained using p-NP acetate as a substrate at different concentrations (0.05 to 4 mM). The kinetic parameters were calculated by analyzing the slopes of the Michaelis-Menten equation using GraphPad Software (GraphPad Inc., USA).

Bacterial strains and plasmids used in this study are summarised in Table 1. Bifidobacteria were routinely cultured on Reinforced Clostridium Agar (RCA) or in modified deMan, Rogosa, Sharpe medium (mMRS) supplemented with 1 % (w/v) lactose (Sigma-Aldrich, Steinheim, Germany) and 0.05% (w/v) cysteine–HCL (Sigma-Aldrich) (Man et al., 1960 (link)). All bifidobacteria were cultivated under anaerobic conditions in a modular atmosphere-controlled system (Davidson and Hardy, Belfast, United Kingdom). Lactococcus lactis strains were grown in M17 broth (Oxoid, Basingstoke, Hampshire, United Kingdom) supplemented with 0.5% (w/v) glucose at 30°C. Where required media was supplemented with 5 mg ml-1 chloramphenicol. For RCA ethyl ferulate plate assays, RCA medium was supplemented with 0.1% (v/v) ethyl ferulate dissolved in 96% ethanol. Methyl ferulate, ethyl ferulate, methyl p–coumaric acid, methyl sinapinate, methyl caffeic acid (caffeate), and feruloyl glucose were all dissolved in 96% ethanol (Carbon Chemicals, Ringaskiddy, Ireland) and sourced from Carbosynth, Berkshire, United Kingdom. Para-nitrophenol (p-Np) acetate, p-Np butyrate, p-Np octanoate and p-Np dodecanoate were purchased from Sigma-Aldrich. All ions were purchased from Sigma-Aldrich.