P np esters

Enzyme activity was determined by colorimetric method measuring released p-nitrophenol from p-nitrophenyl (pNP) esters (Sigma, St. Louis, MO, USA) at 405 nm. Esterase activity was measured by reaction mixture with 1 mM p-nitrophenyl esters in 50 mM Tris–HCl (pH 8.0) containing 1 % (v/v) acetonitrile at 35 °C. The substrate specificity of enzyme was determined in the presence of 1 mM of p-nitrophenyl esters with different aliphatic side chains: acetate (C2), butyrate (C4), hexanoate (C6), octanoate (C8), decanoate (C10), laurate (C12), myristate (C14), palmitate (C16), and stearate (C18). The optimum temperature of enzyme was determined at temperatures ranging from 5 to 70 °C using p-nitrophenyl butyrate as a substrate in 50 mM Tris–HCl buffer (pH 8.0). For optimization of the pH of enzyme, enzyme activity was measured for a pH range of 4.0–10.0. The buffers used were 50 mM sodium acetate (pH 4.0–6.0), 50 mM sodium phosphate (pH 6.0–7.5), 50 mM Tris–HCl (pH 7.5–8.5), and 50 mM CHES (pH 8.5–10.0).

Restriction enzymes were purchased from New England Biolabs (NEB), USA. Taq polymerase and T4 DNA ligase were purchased from Bangalore Genei, India. Gel extraction kit and plasmid isolation kit were purchased from Qiagen, India. Recombinant yeast strain P. pastoris X-33 harbouring Lip11 gene from Yarrowia lipolytica was taken from the laboratory culture collection. This strain has been submitted to Microbial Type Culture Collection (MTCC) with MTCC number 9517. Zeocine was from Invitrogen. The triacylglycerides, p-np esters used in the experiments were procured from Sigma Aldrich. Luria bertani, tryptone, yeast extract, yeast nitrogen base and methanol were purchased from Hi-Media. Sodium chloride was taken from Sisco Research Laboratories Pvt. Ltd. India (SRL). Glycosylation kit was procured from G Bioscience (USA).

p-NP esters with various carbon chain lengths (p-NPC₂ to p-NPC₁₆) were purchased from Sigma-Aldrich (USA) or TCI (Tokyo, Japan). Diisobutyl phthalate (DiBP), dibutyl phthalate (DBP), Bis (2-ethylhexyl) phthalate (DEHP), and diethyl phthalate (DEP) were purchased from J&K Scientific Ltd., China. pEASY-E2 expression kit and fast pfu DNA polymerase were provided by TransGen Biotech (Beijing, China). Escherichia coli BL21(DE3) and pET-28a(+) expression vector was from Novagen (USA). Qiagen provided the Nickel-NTA agarose (Germany). pEGFP-N3, pMD18-T and restriction enzymes BamHI and HindIII were purchased from Takara. Luria–Bertani (LB) bacteria growth medium was obtained from Thermo Fisher Scientific (USA). All other chemicals were at least analytical grade and were obtained from Sigma (USA) or Sinopharm Chemical Reagent (Shanghai, China).