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Biochemistry (Moscow)

, Volume 75, Issue 3, pp 375–381 | Cite as

Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus

  • S. Bansal
  • D. Gnaneswari
  • P. Mishra
  • B. KunduEmail author
Article

Abstract

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K m of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

Key words

Pyrococcus furiosus L-asparaginase hyperthermophile chemical denaturation homology model stability enzyme kinetics 

Abbreviations

AMC

7-amino-4-methylcoumarin

EcAII, ErA, PfA, PhA, TaqA, and TthA

asparaginases from Escherichia coli, Erwinia chrysanthemi, Pyrococcus furiosus, P. horikoshii, Thermus aquaticus, and T. thermophilus

GdnCl

guanidine hydrochloride

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Supplementary material

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Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • S. Bansal
    • 1
  • D. Gnaneswari
    • 1
  • P. Mishra
    • 1
  • B. Kundu
    • 1
    • 2
    Email author
  1. 1.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiHauz Khas, New DelhiIndia
  2. 2.School of Biological SciencesIndian Institute of Technology DelhiHauz Khas, New DelhiIndia

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