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

Selvaraju Gayathri Devi, Anwar Aliya Fathima, Mary Sanitha, Sellamuthu Iyappan, Wayne R. Curtis, Mohandass Ramya

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)694-700
Number of pages7
JournalJournal of Bioscience and Bioengineering
Volume122
Issue number6
DOIs
StatePublished - Dec 1 2016

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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