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Molecular Biotechnology

, Volume 58, Issue 4, pp 256–267 | Cite as

Biochemical and in silico Characterization of Recombinant L-Lactate Dehydrogenase of Theileria annulata

  • Belma Nural
  • Aysegul Erdemir
  • Ozal Mutlu
  • Sinem Yakarsonmez
  • Ozkan Danis
  • Murat Topuzogullari
  • Dilek Turgut-Balik
Original Paper

Abstract

Theileria annulata is a parasite that causes theileriosis in cattle. Reports about drug resistance made essential to develop new drug. LDH of Theileria schizonts is the vital enzyme for its anaerobic metabolism. TaLDH gene was first cloned into pGEM-T cloning vector with two introns in our previous study. Here we report cloning of TaLDH without introns into pLATE 31 vector in E. coli BL21(DE3). Protein was in an inactive form. Two mutations were fixed to express the active protein. Protein was purified by affinity chromatography and evaluated by SDS-PAGE and size exclusion chromatography. Optimum pH of enzyme was performed in pH 7.5, and enzyme was stabilized at 20–40 °C. Enzyme kinetics of recombinant TaLDH were found to be in the direction of pyruvate to lactate K m 0.1324 and K i 4.295 mM, k cat , 44.55/s and k cat /K m , 3.3693 × 105/M/s. 3D structure of TaLDH was predicted, and possible drug binding sites were determined by homology modelling.

Keywords

LDH Theileria annulata Thermostability Substrate inhibition Homology modelling 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Belma Nural
    • 1
  • Aysegul Erdemir
    • 2
  • Ozal Mutlu
    • 3
  • Sinem Yakarsonmez
    • 2
  • Ozkan Danis
    • 4
  • Murat Topuzogullari
    • 2
  • Dilek Turgut-Balik
    • 2
  1. 1.Department of Biotechnology and Biosafety, Institute of ScienceEskisehir Osmangazi UniversityEskisehirTurkey
  2. 2.Department of Bioengineering, Faculty of Chemical and Metallurgical EngineeringYildiz Technical UniversityIstanbulTurkey
  3. 3.Department of Biology, Faculty of Arts and SciencesMarmara UniversityIstanbulTurkey
  4. 4.Department of Chemistry, Faculty of Arts and SciencesMarmara UniversityIstanbulTurkey

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