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 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.
Department of Genetic Engineering, SRM University, Tamilnadu 603203, India.
Selvaraju Gayathri
SG
Devi
Department of Genetic Engineering, SRM University, Tamilnadu 603203, India.
Anwar Aliya
AA
Fathima
Department of Genetic Engineering, SRM University, Tamilnadu 603203, India.
Mary
M
Sanitha
Department of Genetic Engineering, SRM University, Tamilnadu 603203, India.
Sellamuthu
S
Iyappan
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
Wayne R
WR
Curtis
Department of Genetic Engineering, SRM University, Tamilnadu 603203, India. Electronic address: ramya.m@ktr.srmuniv.ac.in.
Mohandass
M
Ramya
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D056186:Metagenomics; D012722:Sewage / Q000382:microbiology*; D013696:Temperature; D013783:Textile Industry
D001426:Bacterial Proteins; D012722:Sewage; D010450:Endopeptidases; C087822:alkaline protease
Activated sludge; Alkaline protease; Enzyme kinetics; Metagenomics; Thermostability