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Identification and characterization of alkaline serine protease from goat skin surface metagenome

Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease...

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Detalles Bibliográficos
Autores principales: Pushpam, Paul Lavanya, Rajesh, Thangamani, Gunasekaran, Paramasamy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159910/
https://www.ncbi.nlm.nih.gov/pubmed/21906326
http://dx.doi.org/10.1186/2191-0855-1-3
Descripción
Sumario:Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co(2+ )or Mn(2+)) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.