Title |
A hydrolytic γ-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation
|
---|---|
Published in |
Extremophiles, October 2012
|
DOI | 10.1007/s00792-012-0490-8 |
Pubmed ID | |
Authors |
Rinky Rajput, Ved Vrat Verma, Vishal Chaudhary, Rani Gupta |
Abstract |
γ-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with γ-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni(2+)-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t (1/2) of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Ptγ-GT shared 37 % sequence identity and 53 % homology with an extremophile γ-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
India | 2 | 15% |
Unknown | 11 | 85% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 6 | 46% |
Researcher | 2 | 15% |
Student > Master | 1 | 8% |
Unknown | 4 | 31% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 4 | 31% |
Biochemistry, Genetics and Molecular Biology | 3 | 23% |
Psychology | 1 | 8% |
Engineering | 1 | 8% |
Unknown | 4 | 31% |