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Extremophiles

, Volume 17, Issue 1, pp 29–41 | Cite as

A hydrolytic γ-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

  • Rinky Rajput
  • Ved Vrat Verma
  • Vishal Chaudhary
  • Rani GuptaEmail author
Original Paper

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 Ni2+-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.

Keywords

Picrophilus torridus Thermo-acidophile γ-Glutamyl transpeptidase Expression PET 51b(+) Site-directed mutagenesis 

Notes

Acknowledgments

Financial assistance from DU/DST-PURSE grant and Misc. R & D Grant, University of Delhi is acknowledged.

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Copyright information

© Springer Japan 2012

Authors and Affiliations

  • Rinky Rajput
    • 1
  • Ved Vrat Verma
    • 1
  • Vishal Chaudhary
    • 1
  • Rani Gupta
    • 1
    Email author
  1. 1.Department of MicrobiologyUniversity of DelhiNew DelhiIndia

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