Mining of a novel esterase (est3S) gene from a cow rumen metagenomic library with organosphosphorus insecticides degrading capability: Catalytic insights by site directed mutations, docking, and molecular dynamic simulations

Int J Biol Macromol. 2021 Nov 1:190:441-455. doi: 10.1016/j.ijbiomac.2021.08.224. Epub 2021 Sep 7.


A novel esterase (est3S) gene, 1026 bp in size, was cloned from a metagenomic library made of uncultured microorganisms from the contents of cow rumen. The esterolytic enzyme (Est3S) is composed of 342 amino acids and shows the highest identity with EstGK1 (71.7%) and EstZ3 (63.78%) esterases from the uncultured bacterium. The Est3S did not cluster in any up-to-date classes (I to XVIII) of esterase and lipase. Est3S protein molecular weight was determined to be 38 kDa by gel electrophoresis and showed optimum activity at pH 7.0 and 40 °C and is partially resistant to organic solvents. Est3S activity was enhanced by K+, Na+, Mg2+, and Ca2+ and its highest activity was observed toward the short-chain p-nitrophenyl esters. Additionally, Est3S can degrade chlorpyrifos (CP) and methyl parathion (70% to 80%) in an hour. A mutated Est3S (Ser132-Ala132) did not show any activity toward CP and ester substrates. Notably, the GHS132QG motif is superimposed with the homolog esterase and cutinase-like esterase. Therefore, Ser132 is the critical amino acid like other esterases. The Est3S is relatively stable with ester compounds, and the methyl parathion complex was confirmed by molecular dynamics simulation. NOVELTY STATEMENT: A novel esterase gene (est3S) expressing esters and organophosphorus insecticide degradation traits was isolated from the uncultured bacterium in the contents of cow rumen. The Est3S protein did not cluster in any up-to-date classes (I to XVIII) of esterase/lipase proteins. Est3S was stable with the ligands up to 100 ns during the molecular dynamic simulations.

Keywords: Insecticides; Metagenomic library; Molecular dynamic simulation; Mutation; Novel esterase (Est3S).

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Biocatalysis
  • Catalytic Domain
  • Cattle
  • Cloning, Molecular
  • Esterases / chemistry
  • Esterases / genetics*
  • Esterases / isolation & purification
  • Esterases / metabolism
  • Gene Library*
  • Kinetics
  • Ligands
  • Metagenomics*
  • Molecular Docking Simulation*
  • Molecular Dynamics Simulation*
  • Molecular Weight
  • Mutagenesis, Site-Directed*
  • Organophosphorus Compounds / metabolism*
  • Phylogeny
  • Physical Chromosome Mapping
  • Recombinant Proteins / metabolism
  • Rumen / enzymology*
  • Substrate Specificity


  • Ligands
  • Organophosphorus Compounds
  • Recombinant Proteins
  • Esterases