P nitrophenyl esters

The plasmid pET‐28a(+) was used to express the target gene in the expression host Escherichia coli BL21(DE3). Restriction endonucleases, DNA polymerase, and T4 DNA ligase were purchased from NEB (New England BioLabs, China). Nickel columns were purchased from GE (General Electric Company, China). p‐Nitrophenyl esters were obtained from Sigma (St. Louis, USA). All other chemicals were of analytical grade and purchased from Sangon (Shanghai, China).

pNP phosphate and all the p-nitrophenyl esters were purchased from Sigma-Aldrich. 4-methylimidazole was purchased from Acros Organics. 1-Isopropoxy-4-nitrobenzene and p-nitrophenyl glycerol were obtained from TCI America and Alfa aesar, respectively. The peptides RADA16H, RADA16H₂, RADA16H₃ and RGDA16H₂ were commercially synthesized form SciLight Biotechnology LLC and used without further purification.

Escherichia coli DH5α (Invitrogen, USA) competent cells were used for transformation and vector amplification. pMD18-T-flip2 (T-vectors harboring full-length YLLip2 gene), plasmid pJME803 (a kind gift from Jean-marc Nicaud, CNRS, Institut Micalis, Domaine de Vilvert, France), and pINA1297 (a kind gift from Catherine Madazak, INRA, UMR1319 Micalis, Domaine de Vilvert, Jouy-en-Josas, France) were stored in our laboratory. Strain Y. lipolytica JMY1212 (a kind gift from Jean-marc Nicaud, CNRS, Institut Micalis, Domaine de Vilvert, France) for protein expression was also stored in our lab. Plasmid extraction kit and DNA purification kit were purchased from Omega (USA). PrimeSTAR HS DNA polymerase, restriction endonucleases, and DNA Ligation Kit were purchased from Takara (Dalian, China). QuikChange Site-Directed Mutagenesis Kit was purchased from Agilent Technologies Inc. (CA, USA). p-Nitrophenyl esters were bought from Sigma-Aldrich (St. Louis, USA). All other reagents used were of analytical grade and commercially available from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

P. synxantha PS1 was isolated from oil well-produced water (Shengli Oilfield, Shandong Province, China). E. coli DH5α (Invitrogen, USA) and plasmid pMD19-T (TaKaRa, Japan) were used for gene cloning and sequencing of AT-containing and AT-truncated carboxylesterases. Plasmid pET-28a (Novagen, USA) was used as a vector to construct the protein expression plasmid in E. coli BL21 (DE3). The substrates fenpropathrin, cypermethrin, fenvalerate, bifenthrin, and p-nitrophenyl esters were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and purchased from local markets.

Esterase activity was determined by measuring the release of p-nitrophenol during the enzymatic hydrolysis of different p-nitrophenyl esters (Sigma-Aldrich). The release of p-nitrophenol was measured at 405 nm using an Ultrospec 2000 pro UV/visible spectrophotometer (Amersham Bioscience). In a total volume of 1.0 mL, esterase was incubated with 0.25 mM p-nitrophenyl butyrate (C₄) as a substrate in 20 mM Tris-HCl buffer (pH 8.17) at 46.27°C for 30 min. One unit of enzyme activity was defined as the amount needed to release 1 μmol p-nitrophenol per min.