Expression and characterization of alkaline protease from the metagenomic library of tannery activated sludge

J Biosci Bioeng. 2016 Dec;122(6):694-700. doi: 10.1016/j.jbiosc.2016.05.012. Epub 2016 Jun 17.


Metagenomics has the potential to facilitate the discovery of novel enzymes; however, to date, only a few alkaline proteases have been characterized from environmentally-sourced DNA. We report the identification and characterization of an alkaline serine protease designated as Prt1A from the metagenomic library of tannery activated sludge. Sequence analysis revealed that Prt1A is closely related to S8A family subtilisins with a catalytic triad of Asp143, His173, and Ser326. The putative protease gene (prt-1A) was subcloned in pET 28a (+) vector and overexpressed in Escherichia coli BL21(DE3)pLysS cells. This 38.8 KDa recombinant protease was purified to homogeneity by nickel affinity chromatography and exhibited optimal enzyme activity at elevated pH (11.0) and temperature (55°C). The enzyme activity was enhanced by the addition of 5 mM Ca2+ ions, and was stable in the presence of anionic detergent, oxidizing agent and various organic solvents. The enzyme displayed high affinity and catalytic efficiency for casein under standard assay conditions (Vmax = 279 U/mg/min, Km = 1.70 mg/mL) and was also compatible with commercial detergents. These results suggest that Prt1A protease could act as an efficient enzyme in various industrial applications.

Keywords: Activated sludge; Alkaline protease; Enzyme kinetics; Metagenomics; Thermostability.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Cloning, Molecular
  • Endopeptidases / genetics*
  • Endopeptidases / metabolism*
  • Enzyme Stability
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Enzymologic
  • Gene Library
  • Hydrogen-Ion Concentration
  • Metagenomics*
  • Sewage / microbiology*
  • Temperature
  • Textile Industry*


  • Bacterial Proteins
  • Sewage
  • Endopeptidases
  • alkaline protease