tailieunhanh - Báo cáo hóa học: " Identification and characterization of alkaline serine protease from goat skin surface metagenome"

Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành hóa học dành cho các bạn yêu hóa học tham khảo đề tài: Identification and characterization of alkaline serine protease from goat skin surface metagenome | Pushpam et al. AMB Express 2011 1 3 http content 1 1 3 o AMB Express a SpringerOpen Journal ORIGINAL ARTICLE Open Access Identification and characterization of alkaline serine protease from goat skin surface metagenome Paul Lavanya Pushpam Thangamani Rajesh Paramasamy Gunasekaran Abstract 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 Co2 or Mn2 for its improved activity. The pH and temperature optima for this protease were and 42 C respectively. Introduction Proteases are present in all living forms as they are involved in various metabolic processes. They are mainly involved in hydrolysis of the peptide bonds Gupta et al. 2002 . Proteases are classified into six types based on the functional groups in their active sites. They are aspartic cysteine glutamic metallo serine and threonine proteases. They are also classified as exo-peptidases and endo-peptidases based on the position of the peptide bond cleavage. Proteases find a wide range of applications in food pharmaceutical leather and textile detergent diagnostics .