P np butyrate

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).

Erythrobacter seohaensis SW-135 was kindly provided by Prof. Jung-Hoon Yoon and cultivated in marine broth 2216 (BD Difco^TM, United States) at 30°C (Yoon et al., 2005 (link)). Kits for genomic DNA isolation, DNA purification, and plasmid isolation were purchased from Omega (United States). DNA polymerase was purchased from TaKaRa (China) and T4 DNA ligase and restriction endonucleases were purchased from New England Biolabs (United States). Plasmid pSMT3 (Herrmann et al., 1996 (link)) was stored in our lab and used as the vector for gene cloning and sequencing as well as protein expression. Escherichia coli BL21 (DE3) was used for protein expression. E. coli strains were grown at 37°C in Lysogeny broth (LB) medium containing 10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract (BD Difco^TM, United States), pH 7.0, supplemented with kanamycin (50 μg/mL) when required. Ni Sepharose (GE Healthcare, United States) was used to purify the His₆-tagged protein. p-Nitrophenyl (NP) acetate (C2), p-NP butyrate (C4), p-NP caprylate (C8), p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), and p-NP palmitate (C16) were purchased from Sigma–Aldrich (United States) and p-NP hexanoate (C6) was purchased from TCI (Japan).