Pnp myristate

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).

Escherichia coli DH5α (Biomed, Beijing, China) was used for propagation of plasmids, and P. pastoris GS115 (his4) was used for protein expression. Trizol reagent (Invitrogen, Carlsbad, USA) and Oligotex mRNA Midi Kit (Qiagen, Dusseldorf, Germany) were used for total RNA extraction and mRNA purification. BD SMART™ RACE cDNA Amplification Kit was purchased from Clontech (Palo Alto, CA, USA). Ex Taq DNA polymerase, PrimeSTAR HS DNA polymerase, restriction endonucleases, and pMD18-T were purchased from TaKaRa (Tokyo, Japan). T4 DNA ligase was purchased from New England Biolabs (Ipswich, MA, USA). The Pichia pastoris expression kit was obtained from Invitrogen (Carlsbad, USA). p-Nitrophenol (pNP), pNP acetate (pNPA), pNP butyrate (pNPB), pNP hexanoate (pNPH), pNP caprylate (pNPC), pNP decanoate (pNPD), pNP laurate (pNPL), pNP myristate (pNPM), and pNP palmitate (pNPP) were purchased from Sigma Chemical Co. (St. Lous, MO, USA). pNP hexanoate (pNPH) was obtained from HEOWNS Company (Tianjin, China). Olive oil, soybean oil, and peanut oil were purchased from a local market. All other chemicals used were of analytical grade unless otherwise stated.

Six p-nitrophenyl (pNP) esters of fatty acids with different chain lengths were obtained from Sigma-Aldrich: pNP acetate (C2), pNP butyrate (C4), pNP decanoate (C10), pNP dodecanoate (C12), pNP myristate (C14) and pNP palmitate (C16). Each enzymatic reaction contained 100 mM sodium phosphate buffer pH 8.0, 4 mg/ml Triton, 0.8 mM of each pNP ester dissolved in acetonitrile:isopropanol mix (80:20 v/v) and 50 nM of purified Est10.
One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 μmol of p-nitrophenol per minute. The production of p-nitrophenol was continuously monitored at 405 nm in a Varioskan Flash (Thermo Scientific) during 15 min at 40°C. The activity of the enzyme was calculated by measuring the initial reaction rate. The data were collected in triplicates and a blank reaction without enzyme was included for each substrate.