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

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 metagen...

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Bibliographic Details
Published in:Journal of bioscience and bioengineering 2016-12, Vol.122 (6), p.694-700
Main Authors: Devi, Selvaraju Gayathri, Fathima, Anwar Aliya, Sanitha, Mary, Iyappan, Sellamuthu, Curtis, Wayne R., Ramya, Mohandass
Format: Article
Language:English
Subjects:
Activated sludge
Alkaline protease
Amino Acid Sequence
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Cloning, Molecular
Endopeptidases - genetics
Endopeptidases - metabolism
Enzyme kinetics
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
Thermostability
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Summary: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.
ISSN:1389-1723
1347-4421
DOI:10.1016/j.jbiosc.2016.05.012